Ⅱ.人类神经突生长的一种抑制蛋白

人们普遍认为成年的中枢神经不能再生[1,2]的原因,和胶质疤痕的空间阻碍作用、促神经生长因子的缺乏及髓鞘抑制因子的存在有关[3-5].这些抑制因子包括:蛋白多糖[6],髓鞘相关的糖蛋白[3,5]以及牛脑中被称为Nogo的两种蛋白[7-9].我们用牛的相关序列获得了人Nogo基因,并分离了其cDNA克隆.蛋白包含三个异构体,对神经突的生长有抑制作用,可能阻碍中枢神经的再生.     

Stimulation of neurite outgrowth by neurotrophins delivered from degradable hydrogels

Degradable hydrogels are useful vehicles for the delivery of growth factors to promote the regeneration of diseased or damaged tissue. In the central nervous system, there are many instances where the delivery of neurotrophins has great potential in tissue repair, especially for treatment of spinal cord injury. In this work, hydrogels based on poly(ethylene glycol) that form via a photoinitiated polymerization were investigated for the delivery of neurotrophins. The release kinetics of these factors are controlled by changes in the network crosslinking density, which influences neurotrophin diffusion and subsequent release from the gels with total release times ranging from weeks to several months. The release and activity of one neurotrophic factor, ciliary-neurotrophic factor (CNTF), was assessed with a cell-based proliferation assay and an assay for neurite outgrowth from retinal explants. CNTF released from a degradable hydrogel above an explanted retina was able to stimulate outgrowth of a significantly higher number of neurites than controls without CNTF. Finally, unique microsphere/hydrogel composites were developed to simultaneously deliver multiple neurotrophins with individual release rates.     

Directional neurite outgrowth is enhanced by engineered meningeal cell-coated substrates

After injury to the CNS, the anatomical organization of the tissue is disrupted, posing a barrier to the regeneration of axons. Meningeal cells, a central participant in the CNS tissue response to injury, migrate into the core of the wound site in an unorganized fashion and deposit a disorganized extracellular matrix (ECM) that produces a nonpermissive environment. Previous work in our laboratory has shown that the presentation of nanometer-scale topographic cues to these cells influences their morphological, cytoskeletal, and secreted ECM alignment. In the present study, we provided similar environmental cues to meningeal cells and examined the ability of the composite construct to influence dorsal root ganglion regeneration in vitro. When grown on control surfaces of meningeal cells lacking underlying topographic cues, there was no bias in neurite outgrowth. In contrast, when grown on monolayers of meningeal cells with underlying nanometer-scale topography, neurite outgrowth length was greater and was directed parallel to the underlying surface topography even though there exists an intervening meningeal cell layer. The observed outgrowth was significantly longer than on laminin-coated surfaces, which are considered to be the optimal substrata for promoting outgrowth of dorsal root ganglion neurons in culture. These results suggest that the nanometer-level surface finish of an implanted biomaterial may be used to organize the encapsulation tissue that accompanies the implantation of materials into the CNS. It furthermore suggests a simple approach for improving bridging materials for repair of nerve tracts or for affecting cellular organization at a device-tissue interface.     

Enhancement of neurite outgrowth in PC12 cells by iron oxide nanoparticles

Despite the many potential therapeutic applications of iron oxide nanoparticle such as its use as an imaging and targeting tool, its biological effects have not yet been extensively characterized. Herein, we report that iron oxide nanoparticles taken up by PC12 cells can enhance neurite outgrowth. PC12 cells exposed to both iron oxide nanoparticles and nerve growth factor (NGF) synergistically increased the efficiency of neurite outgrowth in a dose-dependent manner. This may have resulted from the activation of cell adhesion molecules that are associated with cell-matrix interactions through iron. Immunoblotting assays also revealed that both neural specific marker protein and cell adhesion protein expression were upregulated by iron oxide nanoparticles compared with non-treated cells via activation of the mitogen-activated protein kinase (MAPK) signaling pathway. Our findings point to the possibility that iron oxide nanoparticles can affect cell-substrate interactions and regulate cell behaviors, which provides clinical insights into potential neurologic and therapeutic applications of iron oxide nanoparticles.     

Nervous system proteoglycans as modulators of neurite outgrowth

The proteoglycans are multifunctional macromolecules composed of a core polypeptide and a variable number of glycosaminoglycan chains. The structural diversity and complexities of proteoglycan expression in the developing and adult Nervous System underlies the variety of biological functions that these molecules fulfill. Thus, in the Nervous System, proteoglycans regulate the structural organisation of the extracellular matrix, modulate growth factor activities and cellular adhesive and motility events, such as cell migration and axon outgrowth. This review summarises the evidences indicating that proteoglycans have an important role as modulators of neurite outgrowth and neuronal polarity. Special emphasis will be placed on those studies that have shown that proteoglycans of certain subtypes inhibit neurite extension either during the development and/or the regeneration of the vertebrate Central Nervous System.     

Stimulation of neurite outgrowth using positively charged hydrogels

Autologous nerve grafts are currently the best option for the treatment of segmental peripheral nerve defects. However, autografts have several drawbacks including size mismatch and loss of sensation in the donor nerve's sensory distribution. In this work, we have investigated the development of a synthetic hydrogel that contains positive charge for use as a substrate for nerve cell attachment and neurite outgrowth in culture. We have demonstrated that modification of oligo-(polyethylene glycol) fumarate (OPF) with a positively charged monomer improves primary sensory rat neuron attachment and differentiation in a dose-dependent manner. Positively charged hydrogels also supported attachment of dorsal root ganglion (DRG) explants that contain sensory neurons, Schwann cells and neuronal support cells. Furthermore, charged hydrogels were analyzed for the appearance of myelinated structures in a co-culture containing DRG neurons and Schwann cells. DRGs and Schwann cells remained viable on charged hydrogels for a time period of three weeks and neurites extended from the DRGs. Sudan black staining revealed that neurites emerging from DRGs were accompanied by migrating Schwann cells. These findings suggest that charged OPF hydrogels are capable of sustaining both primary nerve cells and the neural support cells that are critical for regeneration.     

Role of the Rho GTPase-activating protein RICS in neurite outgrowth

The Rho family of small GTPases, including RhoA, Rac1 and Cdc42, are critical regulators of the actin cytoskeleton. In neuronal systems, Rho GTPase-activating proteins (RhoGAPs) and their substrates, Rho GTPases, have been implicated in regulating multiple processes in the morphological development of neurons, including axonal growth and guidance, dendritic elaboration and formation of synapses. RICS is mainly expressed in the brain and functions as a RhoGAP protein for Cdc42 and Rac1 in vitro. To examine the biological function of RICS, we disrupted the RICS gene in mice. RICS knockout mice developed normally and were fertile. However, when cultured in vitro, Cdc42 activity in RICS(-/-) neurons was higher than that in wild-type neurons. Consistent with this finding, hippocampal and cerebellar granule neurons derived from RICS(-/-) mice bore longer neurites than those from wild-type mice. These findings suggest that RICS plays an important role in neurite extension by regulating Cdc42 in vivo.     

Effect of scoparone on neurite outgrowth in PC12 cells

The neurite outgrowth-promoting effects of scoparone isolated from the stem bark of Liriodendron tulipifera were investigated in PC12 cells. At a concentration of 200 microM, scoparone markedly induced neurite outgrowth from PC12 cells. Scoparone at 200 microM also enhanced the outgrowth of neurites from cells in the presence of nerve growth factor (NGF, 2 ng/ml). The levels of intracellular cyclic AMP and concentration of Ca2+ were also increased by 200 microM scoparone. In addition, scoparone at 200 microM increased the activities of extracellular signal-regulated protein kinase (ERK), cyclic AMP-dependent protein kinase (PKA), protein kinase C (PKC) and Ca2+/calmodulin kinase II (CaMK II). However, scoparone-induced neurite outgrowth was blocked by a mitogen-activated protein kinase inhibitor (U0126), a PKA inhibitor (H89), a PKC inhibitor (GF109203X) and a CaMK II inhibitor (KN62). These kinase inhibitors also reduced the scoparone-induced neurite outgrowth associated with NGF. These results suggest that scoparone can induce neurite outgrowth by stimulating the upstream steps of ERK, PKA, PKC and CaMK II in PC12 cells.     

Effects of extracellular calcium concentration on neurite outgrowth in PC12 cells by staurosporine

Staurosporine as an inhibitor of protein kinases can induce neuronal differentiation in PC12 cells. We investigated the role of extracellular Ca²⁺ on staurosporine inducing neurite outgrowth in PC12 cells. The cells were cultured during cell differentiation in the presence of 214 nM staurosporine with 0.0–0.7 Ca²⁺mM as treatment media. We obtained the fraction of neurite-bearing cells, total neurite length and the percentage of cytotoxiciy. The results showed that decrease or increase of extracellular calcium ions resulted in correspondingly significant decrease or increase in total neurite length and cell differentiation in treated cells. With an increase of extracellular calcium concentration from 0.0 to 0.7 mM, the percentage of cytotoxicity of the PC12 cells decreased (p < 0.05). Our data suggest that staurosporine uses the extracellular calcium ions to affect on neurite outgrowth.     

Effects of serotonin on neurite outgrowth from thalamic neurons in vitro

Altering levels of serotonin in the primary somatosensory cortex during early postnatal life influences thalamocortical development. Recent in vivo experiments suggest that serotonin may have direct effects on the growth of thalamocortical axons, and the present study was undertaken to determine whether this amine influences process outgrowth from thalamic cells maintained in culture. Ventrobasal thalamic neurons were harvested from newborn rats and maintained in culture for eight days. At the end of this period, 0, 10, 25, 50 or 100 microM serotonin was added to the culture medium. After an additional six days, cultures were fixed and stained with neuron-specific enolase. Quantitative analysis of >500 cells from each condition indicated that 25 microM serotonin, but not the other concentrations of this amine, significantly increased the length of the primary (longest) process growing out from the cell body (P < 0.001), the total (summed) length of all processes (P < 0.0001), total neurites per cell (P < 0.05), number of branch points per cell (P < 0.01) and branch points on the primary neurite (P < 0.0005). These results demonstrate that exposing thalamic cells to serotonin increases process outgrowth from them in the absence of their cortical targets.     

Induction of neurite outgrowth in PC12 cells by the medium-chain fatty acid octanoic acid

It has been shown that polyunsaturated fatty acids such as arachinonic and docosahexanoic acids but not monounsaturated and saturated long-chain fatty acids promote basal and nerve growth factor (NGF)-induced neurite extension of PC12 cells, a line derived from a rat pheochromocytoma. On the other hand, short-chain fatty acids and valproic acid (2-propylpentanoic acid) enhance the growth of neurite processes of the cells only in the presence of inducers. In this study, we demonstrated that straight medium-chain fatty acids (MCFAs) at millimolar concentrations alone potently induced neuronal differentiation of PC12 cells. Hexanoic, heptanoic and octanoic acids dose-dependently induced neurite outgrowth of the cells: their maximal effects determined 2 days after addition to the culture medium were more marked than the effect of NGF. PC12 cells exposed to octanoic acid expressed increased levels of the neuronal marker beta-tubulin isotype III. Nonanoic, decanoic, and dodecanoic acids also induced growth of neurite processes, but their maximal effects were less marked than that of octanoic acid. In contrast, the polyunsaturated fatty acid linoleic acid and short-chain fatty acids had only slight or almost no effects on neurite formation in the absence of NGF. The effect of octanoic acid was synergistic with or additive to the effects of NGF and dibutyryl cyclic AMP. Octanoic acid upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), critical signaling molecules in neuronal differentiation, but not phosphorylation of Akt, a signaling molecule downstream of phosphatidylinositol 3-kinase (PI3K). Moreover, growth of neurites induced by octanoic acid was potently inhibited by treatment of cells with the p38 MAPK inhibitor SB203580 and the ERK kinase inhibitor PD98059 but not inhibited and only slightly inhibited by the JNK inhibitor SP600125 and the PI3K inhibitor wortmannin, respectively. Taken together, our results indicate that MCFAs, including octanoic acid, induced neurite outgrowth of PC12 cells in the absence of NGF and suggest that the activation of p38 MAPK and ERK pathways is involved in this process.     

Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth.

Thy-1 monoclonal antibodies (MAbs) have previously been shown to promote neurite outgrowth from retinal ganglion cells and a variety of other neurons. We have studied the effect on neurite outgrowth of several Thy-1 MAbs with quantitatively similar binding properties and found that only certain Thy-1 MAbs promote neurite outgrowth. This finding suggests that the antibody effects depend on specific interactions with one or more active sites on the Thy-1 glycoprotein.     

Immobilized concentration gradients of nerve growth factor guide neurite outgrowth

Axons are guided to their targets by a combination of haptotactic and chemotactic cues. We previously demonstrated that soluble neurotrophic factor concentration gradients guide axons in a model system. In an attempt to translate this model system to a device for implantation, our goal was to immobilize a stable neurotrophic concentration gradient for axonal (or neurite) guidance. Nerve growth factor (NGF) was immobilized within poly(2-hydroxyethylmethacrylate) [p(HEMA)] microporous gels using a gradient maker. The NGF was stably immobilized, with only approximately 0.05% of the amount originally incorporated into the gel released over an 8-day period. Immobilized NGF was bioactive: the percent of PC12 cells extending neurites on NGF-immobilized p(HEMA) gels was 16 +/- 2%, which was statistically the same as those exposed to soluble NGF (22 +/- 6%). We were able to predict and reproducibly create stable NGF concentration gradients in the gel. At an NGF concentration gradient of 357 ng/mL/mm, PC12 cell neurites were guided up the gradient. The facile, flexible, and reproducible nature of this method allowed us to translate soluble growth factor gradient models to stable growth factor gradient devices that may ultimately enhance axonal guidance and regeneration in vivo.     

Polygalasaponin G promotes neurite outgrowth of cultured neuron on myelin

Myelin contains many axonal outgrowth inhibitory components which contribute to regeneration failure after neuronal injury in the mammalian central nervous system (CNS). In an attempt to develop small molecular agents to promote axonal outgrowth, we screened a compound library purified from traditional Chinese herbs, and found a small molecular compound polygalasaponin G (PS-G), extracted from Polygala japonica, which has a potent neurotrophic activity on PC12 cells and cultured cortical neurons. We reported, to our knowledge for the first time, that PS-G could promote neurite outgrowth of neurons cultured on the myelin substrates and inhibit the activation of RhoA. Thus, our results could represent a therapeutic approach to improve axon regeneration after CNS injuries.     

Ndrg2 promotes neurite outgrowth of NGF-differentiated PC12 cells

Ndrg2 is a member of the N-myc downstream-regulated genes. Thus far, two different isoforms of rat Ndrg2 protein, Ndrg2S and Ndrg2L, have been identified. Recently, we have identified rat Ndrg2 as a novel target molecule of antidepressants and ECT. The functional role of Ndrg2 in the central nervous system, however, remains unclear. In the present study, we examined the expression of endogenous Ndrg2, cellular localization of transfected Ndrg2 protein, and morphological changes resulting from overexpression of Ndrg2 in NGF-differentiated PC12 cells. Neurites began to sprout 1-2 days after exposure to NGF; subsequent neurite growth continued for 5 days. During this time, we evaluated Ndrg2 mRNA expression by real-time quantitative PCR and found that expression significantly increased in a time-dependent manner. Interestingly, V5-conjugated Ndrg2S and Ndrg2L proteins expressed in NGF-differentiated PC12 specifically localized to cell surface membranes and growth cones. Moreover, Ndrg2S and Ndrg2L overexpression promoted neurite elongation in NGF-differentiated PC12 cells. In conclusion, our findings offer novel insights into the physiological roles of Ndrg2 in the central nervous system.     

Enhancement of neurite outgrowth using nano-structured scaffolds coupled with laminin

Cell interactions with scaffolds are important for cell and tissue development in the process of repairing and regeneration of damaged tissue. Scaffolds that mimic extracellular matrix (ECM) surface topography, mechanical stiffness, and chemical composition will be advantageous to promote enhanced cell interactions. Electrospinning can easily produce nano-structured synthetic polymer mats with architecture that structurally resembles the ECM of tissue. Although electrospinning can produce sub-micron fibrous scaffolds, modification of electrospun scaffolds with bioactive molecules is beneficial as this can create an environment that consists of biochemical cues to further promote cell adhesion, proliferation and differentiation. Incorporation of laminin, a neurite promoting ECM protein, onto the nanofibers is an alternative to further mimic the biochemical properties of the nervous tissue to create a biomimetic scaffold. In this study, we investigated the feasibility to functionalize scaffolds by coupling laminin onto poly(L-lactic acid) (PLLA) nanofibers. Laminin was successfully added to nanofibers using covalent binding, physical adsorption or blended electrospinning procedures. PC12 cell viability and neurite outgrowth assays confirmed that the functionalized nanofibers were able to enhance axonal extensions. Significantly, compared to covalent immobilization and physical adsorption, blended electrospinning of laminin and synthetic polymer is a facile and efficient method to modify nanofibers for the fabrication of a biomimetic scaffold. Using these functionalization techniques, nanofibers can be effectively modified with laminin for potential use in peripheral nerve regeneration applications.     

The neurite outgrowth inhibitor Nogo A is involved in autoimmune-mediated demyelination

Inhibitors associated with CNS myelin are thought to be important in the failure of axons to regenerate after spinal cord injury and in other neurodegenerative disorders. Here we show that targeting the CNS-specific inhibitor of neurite outgrowth Nogo A by active immunization blunts clinical signs, demyelination and axonal damage associated with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). Mice vaccinated against Nogo A produce Nogo-specific antibodies that block the neurite outgrowth inhibitory activity associated with CNS myelin in vitro. Passive immunization with anti-Nogo IgGs also suppresses EAE. Our results identify Nogo A as an important determinant of the development of EAE and suggest that its blockade may help to maintain and/or to restore the neuronal integrity of the CNS after autoimmune insult in diseases such as MS. Our finding that Nogo A is involved in CNS autoimmune demyelination indicates that this molecule may have a far more complex role than has been previously anticipated.